Millions of patients in the US and worldwide are affected by chronic wounds which resulting in billions of dollars of expenditure every year in patient care. While extensive efforts have been placed on the development of new therapies to improve wound healing, little progress has been made thus far on wound diagnosis and monitoring. Currently, recording wound area and volume is routinely done as the major means of determining progress in wound healing. The wound area measurement techniques are very subjective, time-consuming and imprecise. A new wound monitoring technology is urgently needed for assessing wound healing status which is essential for effective wound care and improving patient outcomes. Reactive oxygen species (ROS) is well established as a sensitive biomarker of wound healing. While ROS has been shown to promote cell proliferation and migration, excessive and low ROS have been documented to have a negative impact on wound healing. This Phase II SBIR study is aimed at developing a new non-invasive method for assessing wound healing status by quantitating and mapping ROS activity in the wounds in real-time. Several ROS-reactive bioluminescent probes have been investigated in recent years to detect ROS activities in vivo. Unfortunately, these probes alone cannot be used to quantify the degree of ROS activities and inflammatory responses due to the fact that the extent of the bioluminescent signals is also probe-concentration dependent. To address this challenge, we adopt ratiometric probe technique in which both ROS-sensitive chemiluminescent agents and ROS-insensitive fluorescent dye as reference are conjugated to biocompatible particle carriers. The bioluminescence/reference fluorescence intensity ratios can be calculated to reflect the extent of localized ROS activities while circumventing the variations of bioluminescence intensities associated with the ROS probe concentrations. Our Phase I studies have led to the development of ratiometric ROS probes which can reliably detect localized ROS activities in vivo and inflamed tissue in the wound site. Based on these exciting observations, the proposed work will optimize the ratiometric ROS probe fabrication technique and synthesize ratiometric ROS probe-conjugated wound dressing sheets. By placing on top of chronic wounds, the dressing will adsorb exudate from the wound which contains a lot of inflammatory cells and ROS products. The incoming ROS products will react with the ratiometric probes nearby to provide real time and continual assessment of inflammatory responses and healing activity at different areas of the wounds. For the development of such technology, we will first evaluate the ability of both ROS probe-conjugated wound dressing sheets to detect ROS activities in vitro (Aim 1). Since all commercial imagers are designed with an enclosed chamber only suitable for cell and small animal imaging, a handheld imager and software platform for ROS sensing in large animals and humans will be developed as part of this investigation (Aim 2). Finally, the ability of probe- conjugated wound dressings, and handheld imager system to assess acute, chronic and infected wound will be evaluated using porcine skin wound models (Aim 3). The successful completion of the proposed work will help in the development of a new monitoring tool for wound care. Equally importantly, the proposed work will provide critical data that is needed for seeking FDA approval and commencing human studies.